CN102988970B - Foot-and-mouth disease purification vaccine, preparation method and applications thereof - Google Patents
Foot-and-mouth disease purification vaccine, preparation method and applications thereof Download PDFInfo
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Abstract
The present invention discloses a foot-and-mouth disease purification vaccine, a preparation method and applications thereof. The preparation method comprises: adopting protamine sulfate precipitation to remove most of hybridproteins and nucleic acids of host cells; condensing a virus liquid through a concentration system to be adopted as a chromatography sample; and selecting a polyacrylamide dextran gel as a chromatography medium to carry out purification elution, collecting a first elution peak, carrying out filtration sterilization inactivation, adding an adjuvant to carry out emulsification, and carrying out sub-packaging to obtain the foot-and-mouth disease purification vaccine. The preparation method has characteristics of economy, efficiency, low cost, simple and reasonable process, and effective removal of impurities in the host cells and the culture medium, wherein virus recovery rate is more than 85%, protein and nucleic acid removal rate is more than 90%, and the foot-and-mouth disease purification vaccine prepared by the preparation method has characteristics of safety, good immunity and stable property.
Description
Technical field
The present invention relates to a kind of foot-and-mouth disease vaccine and preparation method thereof, the foot and mouth disease purified vaccine that is particularly related to a kind of preparation method of foot and mouth disease purified vaccine and is obtained by this preparation method, also relates to the application of the foot-and-mouth disease vaccine obtaining in preparation prevention animal foot and mouth disease medicine.Belong to vaccine preparation field.
Background technology
Along with the raising of domestic animal intensive culture level, especially the increase of raising dairy cattle amount, the slight side reaction of vaccine, just can bring huge economic loss, as degradation under conceived cattle miscarriage, the decline of cow in milk milk yield, milk-quality, on market, there is huge demand space without the vaccine of any side reaction.
Mainly there are following two problems in existing inactivated foot-and-mouth disease vaccine: 1. immune protective efficiency is unstable, is mainly manifested in: after immunity, can't detect neutralizing antibody or antibody horizontal very low, or duration of immunity is shorter, only has 2～3 months; 2. immune side reaction is large, mainly: downstream production technology is still traditional method, without purification process, have impurity and anaphylaxis material in some culture medium and host cell, and Effective Antigens content is low in vaccine.These immune prevention and control on foot and mouth disease bring great impact.
In the last few years, also rose about the research heroes of vaccine separation and purification both at home and abroad, be a dark horse, made brilliant achievements.Use in biological product people, the vaccine authenticating by FDA has hundreds of, and the vaccine of developing reaches thousands of kinds more than, about scientific paper and the patent of vaccine research nearly all relate to separating and purifying technology, this class document presents Exponential growth situation in recent years.But separating and purifying technology is applied not yet in the production of live vaccine.Along with the development of China's animal husbandry, people propose requirements at the higher level to live vaccine purity and safety etc.
The separation and purification of vaccine is mainly the foreign protein of removing in culture medium and host cell, peptide, the endotoxin of nucleic acid and contaminated bacteria and its generation, traditional sucrose density gradient centrifugation, precipitation and filtering technique are still generally adopted in the antigen purification of vaccine, but the rough vaccine that the overwhelming majority is simple separation to be obtained, wherein antigenic content is lower, and may contain some impurity and anaphylaxis material, generally all there is purity in the processing method of these traditional vaccines, vigor and safety are on the low side, be not suitable for being applied to the shortcomings such as large-scale production, can not meet the needs of the research and production of novel vaccine.
The purification of vaccine is purified the most reliable with chromatography, the field of application only limits to people's biological product at present, in the research of a handful of viral vaccines such as hepatitis B, mad dog, hemorrhagic fever, influenza, poliomyelitis vaccine and producing.At home and abroad there is no and carry out the report that live vaccine is purified with chromatographic technique.
The breakthrough of this project invention technology, not only improve China's animal vaccine production technology level and vaccine quality, promote the development of China's live vaccine industry, and will impel China's development of making the country prosperous from production of vaccine big country to production of vaccine, conform with world's bio-pharmaceuticals development trend, have broad application prospects.
Summary of the invention
First technical problem to be solved by this invention is to provide a kind of method of preparing foot and mouth disease purified vaccine, the preparation method of a kind of foot and mouth disease purified vaccine of the present invention, it is characterized in that comprising: by after the cell debris in the hoof-and-mouth disease venom of cell suspension cultures is removed, be concentrated into suitable multiple, with the protamine sulfate precipitation most of host cell foreign protein of removal and nucleic acid, centrifugal, supernatant carries out eluting by Sephacryl gel filtration chromatography, collect first eluting peak, deactivation after filtration sterilization, add adjuvant to carry out emulsifying, subpackage and get final product.
Preparation method of the present invention, is characterized in that concrete comprising the following steps:
(1) by the BHK21 cell of hoof-and-mouth disease seed culture of viruses poison inoculation suspension culture, continue suspension culture 15～28 hours;
(2) results, through the foot and mouth disease virus of BHK21 cell suspension cultures, are removed cell debris by microporous filter system, obtain micro-filtrate;
(3) micro-filtrate is concentrated into the 1/10-1/100 of micro-filtrate original volume, obtains the concentrated solution of micro-filtrate;
(4) in the concentrated solution obtaining to step (3), add protamine sulfate, make the final concentration of protamine sulfate reach 1.0mg/ml～2.5mg/ml's, room temperature (25 DEG C) stirs 0.5h～2.0h, leave standstill after effect 0.5h～2.0h, centrifugal 15min～the 60min of 5000rpm～10000rpm, abandon precipitation, get centrifugal supernatant, obtain concentrated foot and mouth disease virus supernatant;
(5) pack polyacrylamide Portugal gel into chromatographic column, with this chromatographic column of phosphate buffer balance of the 0.01M of PH7.4～8.0 of 1～2 times of column volume, the concentrated foot and mouth disease virus supernatant of the centrifugal acquisition of step (4) is added on above this Sephacryl gel, after treating that virus liquid all enters gel, then carry out eluting with the 0.01M phosphate buffer of PH7.4～8.0;
(6) collect first eluting peak, after filtration sterilization with 30 DEG C of deactivations of BEI of 1mM～3mM 28 hours, deactivation liquid (is used the 0.01M phosphate buffer dilution of PH7.4～8.0 after 2～10 times of dilutions, on the books in specific embodiment), add mineral oil adjuvant emulsion by 1: 1 weight ratio, subpackage obtains foot and mouth disease purified vaccine.
In the present invention, described hoof-and-mouth disease poison strain be the each serogroup vaccine strain of foot and mouth disease virus wherein at least one.This be because the column chromatography purification method of foot and mouth disease virus of the present invention according to being complete foot and mouth disease virus particle size, and the complete foot and mouth disease virus particle size of the various strain of foot and mouth disease virus is the same, thereby the various serogroup vaccine strains of foot and mouth disease virus all can adopt method of the present invention to carry out the preparation of purified vaccine.
In specific embodiments of the invention, described hoof-and-mouth disease poison strain is the O type foot and mouth disease seed culture of viruses ONXC/92 strain (foundation (I) of foot and mouth disease virus O type-Asia I type bivalent inactivated vaccine seed culture of viruses seed lot, Xu Chunhe etc., the ten scientific seminar's meeting paper of China's animal and veterinary association's foot and mouth disease credit meeting), Asia I type seed culture of viruses Asia1/JSL/GSZY/06 strain (foot and mouth disease O type, development and the application of Asia I type bivalent inactivated vaccine, Huang Jiong, veterinary's guide, 06 phase in 2009), (Schweineseuche O-shaped inactivated vaccine PD (50) effect inspection counteracting toxic substances mode is explored in O type foot and mouth disease seed culture of viruses OR/80 strain, Lang Hongwu etc., " Chinese veterinary drug magazine " 03 phase in 2004) or O type foot and mouth disease seed culture of viruses O/ZK/93-08 strain (Schweineseuche O-shaped inactivated vaccine OZK/93 strain and OZK/93 strain+OS/99 strain immune effect contrast test, Xu Xinhong etc., feedstuff and herding scale raise pigs 2010, (9)).Described seed culture of viruses---O type foot and mouth disease seed culture of viruses ONXC/92 strain, Asia I type seed culture of viruses Asia1/JSL/GSZY/06 strain, the seed culture of viruses OR/80 strain of O type foot and mouth disease or O type foot and mouth disease seed culture of viruses O/ZK/93-08 strain are bought from Lanzhou veterinary institute.
In specific embodiments of the invention, described microporous filter optimum system choosing aperture is 5 microns, combine use with together with the filter element of 0.45 micron for 1 micron, or 5 microns, combine use with together with the vibrosieve of 0.45 micron for 1 micron, or 5 microns, combine use for 1 micron together with 0.45 micron of hollow fiber column.
In specific embodiments of the invention, it is concentrated or use the film bag that hollow fiber column that molecular cut off is 6kd-20kd or molecular cut off are 6kd-20kd to concentrate that simmer down to described in step (3) uses PEG6000 to carry out virus precipitation.
In specific embodiments of the invention, described Sephacryl gel is that component capture range is 1.0 × 10
4～1.0 × 10
8the high-resolution Sephacryl gel of globulin molecule amount.More preferably component capture range is 1.0 × 10
4～1.5 × 10
6the high-resolution Sephacryl gel of globulin molecule amount.Described Sephacryl gel includes but not limited to sephacryl S-300, sephacryl S-400 or sephacryl S-500.More preferably sephacryl S-300.
Protamine sulfate (article No. p4380), BEI, is sigma company product; 206 adjuvants are purchased from French Seppic company; PEG6000 is purchased from AMRESCO company.Void column XK26; Gel media filler: sephacryl S-300, sephacryl S-400 and sephacryl S-500 are GE company product.
Second technical problem to be solved by this invention is to provide the foot and mouth disease purified vaccine being prepared by above-described preparation method.
The 3rd technical problem to be solved by this invention is to provide the application in preparation prevention animal foot and mouth disease disease medicament of foot and mouth disease purified vaccine that the inventive method prepares.
The preparation method of foot and mouth disease purified vaccine of the present invention, by foot and mouth disease virus being inoculated to the BHK21 cell of suspension culture, after results, remove cell debris, after suitably concentrating, with the protamine sulfate precipitation most of host cell foreign protein of removal and nucleic acid, centrifugal, get centrifugal supernatant as chromatography loading sample, chromatography media Sephacryl gel economical and efficient, treating capacity is large, chemical stability is high, better tolerance, being applicable to very much industrialization uses, and cost is low, technique is simple, rationally, can effectively remove the impurity in host cell and culture medium, foreign protein and nucleic acid clearance are greater than 90%, the virus response rate is greater than 85%, the foot and mouth disease purification inactivated vaccine safety being prepared by the inventive method, immunity is good, stable in properties.
Brief description of the drawings
Fig. 1 is the chromatography collection of illustrative plates that uses Sepharose 4FF to obtain;
Fig. 2 is the chromatography collection of illustrative plates that uses Sepharose 6FF to obtain;
Fig. 3 is the chromatography collection of illustrative plates that uses DEAE Sepharose to obtain;
Fig. 4 is the chromatography collection of illustrative plates that uses Sephacryl S-500 to obtain;
Fig. 5 is the chromatography collection of illustrative plates that uses Sephacryl S-400 to obtain;
Fig. 6 is the chromatography collection of illustrative plates that uses Sephacryl S-300 to obtain.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation of 1 N of foot and mouth disease bivalence deactivation purified vaccine of embodiment (ONXC strain+JSL strain)
1 material
1.1 vaccine strains: manufacturing this product O type inactivated foot-and-mouth disease vaccine production seed culture of viruses is that ONXC/92 strain and the production of Asia I type inactivated foot-and-mouth disease vaccine are that Asia1/JSL/GSZY/06 strain is bought from Lanzhou veterinary institute with seed culture of viruses.
1.2 chemical reagent: BHK21 cell is prepared according to existing conventional method for this experiment, preservation; Protamine sulfate (article No. p4380), BEA, is sigma company product; 206 adjuvants are purchased from French Seppic company.
1.3 chromatographic columns and gel media: gel media filler sephacryl S-300, void column XK26 are GE company product;
1.4 microporous filter systems: aperture is 5 microns, combine use, Shanghai Jin Ke Filters company limited product with together with the filter element of 0.45 micron for 1 micron.
1.5 concentration systems: the film bag that molecular cut off is 10kd, Sartorius AG manufactures.
2 methods
(1) hoof-and-mouth disease seed culture of viruses poison ONXC/92 strain, Asia1/JSL/GSZY/06 strain are inoculated respectively to the BHK21 cell of suspension culture, continued to cultivate 20 hours.
(2) gather in the crops respectively foot and mouth disease virus ONXC/92, the Asia1/JSL/GSZY/06 through BHK21 cell suspension cultures, it is 5 microns by aperture respectively, the filter element of 1 micron and 0.45 micron is removed cell debris, obtains the micro-filtrate of foot and mouth disease virus ONXC/92, Asia1/JSL/GSZY/06.
(3) micro-filtrate that step (2) obtains is concentrated into 1/40 of original volume by film bag ultrafiltration and concentration system respectively.
(4) in the ultrafiltration and concentration liquid of the micro-filtrate obtaining to step (3) respectively, add protamine sulfate, the final concentration that makes protamine sulfate is 1.0mg/ml, stirring at room temperature 0.5h, leave standstill after effect 1h, the centrifugal 30min of 7000rpm, abandons precipitation, gets centrifugal supernatant.
(5) pack sephacryl S-300 gel into chromatographic column, with the phosphate buffer balance chromatographic column of the 0.01M of PH7.4～8.0 of 2 times of column volumes, these two kinds concentrated hoof-and-mouth disease venom of centrifugal acquisition are added on respectively above sephacryl S-300 gel separately, after treating that virus liquid all enters gel, then carry out eluting with the 0.01M phosphate buffer of PH7.4～8.0.
(6) collect respectively first eluting peak, (BEA adds cyclisation under the effect of sodium hydroxide of 0.2N and generates BEI after filtration sterilization, to use respectively the BEI of 3mM.) 30 DEG C of deactivations 28 hours, deactivation ONXC/92 strain and Asia1/JSL/GSZY/06 strain antigen liquid mix by 1: 1 volume ratio, carry out after 4 times of dilutions with the 0.01M phosphate buffer of PH7.4～8.0, add 206 adjuvant emulsions by 1: 1 weight ratio again, subpackage obtains cattle foot and mouth disease bivalence deactivation purified vaccine.
The preparation of the Schweineseuche O-shaped deactivation purified vaccine of embodiment 2 (OR/80 strain+O/ZK/93-08 strain)
1 material
1.1 vaccine strains: manufacturing this product foot and mouth disease seed culture of viruses is that the production seed culture of viruses OR/80 strain of O type inactivated foot-and-mouth disease vaccine and O type inactivated foot-and-mouth disease vaccine production seed culture of viruses O/ZK/93-08 strain are bought from Lanzhou veterinary institute.
1.2 chemical reagent: BHK21 cell is prepared according to existing conventional method for this experiment, preservation; Protamine sulfate (article No. p4380), BEA, is sigma company product; 206 adjuvants are purchased from French Seppic company.
1.3 chromatographic columns and gel media: void column XK26; Gel media filler: sephacryl S-300 is GE company product;
1.4 microporous filter systems: aperture is 5 microns, combine use, Tianjin Ai Sheng membrane filtration technique company limited product with together with the hollow fiber column of 0.45 micron for 1 micron.
1.5 concentration systems: the hollow fiber column that molecular cut off is 6-10kd, Tianjin Ai Sheng membrane filtration technique company limited product.
2 methods
(1) the OR/80 strain of hoof-and-mouth disease seed culture of viruses poison and O/ZK/93-08 strain are inoculated respectively to the BHK21 cell of suspension culture, continued to cultivate 25 hours.
(2) results are through the foot and mouth disease virus of BHK21 cell suspension cultures respectively, and the hollow fiber column that is 5 microns, 1 micron and 0.45 micron by aperture is removed cell debris, obtains respectively the micro-filtrate of hoof-and-mouth disease seed culture of viruses poison OR/80 and O/ZK/93-08.
(3) micro-filtrate that step (2) obtains is concentrated into 1/60 of original volume with hollow fiber column ultrafilter concentration systems respectively.
(4) in the ultrafiltration and concentration liquid of the micro-filtrate obtaining to step (3) respectively, add protamine sulfate, the final concentration that makes protamine sulfate is 2.0mg/ml, stirring at room temperature 1.5h, leave standstill after effect 0.5h, the centrifugal 15min of 10000rpm, abandons precipitation, gets centrifugal supernatant.
(5) pack sephacryl S-300 into chromatographic column, with the phosphate buffer balance chromatographic column of the 0.01M of PH7.4～8.0 of 2 times of column volumes, the concentrated foot and mouth disease virus centrifuged supernatant of centrifugal acquisition is added on respectively above gel separately, after treating that virus liquid all enters gel, then carry out eluting with the 0.01M phosphate buffer of PH7.4～8.0.
(6) collect respectively first eluting peak, (BEA adds cyclisation under the effect of sodium hydroxide of 0.2N and generates BEI after filtration sterilization, to use respectively the BEI of 2.0mM.) 30 DEG C of deactivations 28 hours, deactivation OR/80 strain and O/ZK/93-08 strain antigen mix by 1: 1 volume ratio, carry out after 5 times of dilutions with the 0.01M phosphate buffer of PH7.4～8.0, then add 206 adjuvant emulsions by 1: 1 weight ratio, subpackage obtains Schweineseuche O-shaped deactivation purified vaccine.
The preparation of the Schweineseuche O-shaped deactivation purified vaccine of embodiment 3 (OR/80 strain+O/ZK/93-08 strain)
1 material
1.1 vaccine strains: manufacturing this product foot and mouth disease seed culture of viruses is that the production seed culture of viruses OR/80 strain of O type inactivated foot-and-mouth disease vaccine and O type inactivated foot-and-mouth disease vaccine production seed culture of viruses O/ZK/93-08 strain are bought from Lanzhou veterinary institute.
1.2 chemical reagent: BHK21 cell is prepared according to existing conventional method for this experiment, preservation; Protamine sulfate (article No. p4380), BEA, is sigma company product; 206 adjuvants are purchased from French Seppic company; PEG6000 is purchased from AMRESCO company.
1.3 chromatographic columns and gel media: void column XK26; Gel media filler: sephacryl S-300 is GE company product;
1.4 microporous filter systems: aperture is 5 microns, combine use, Tianjin Ai Sheng membrane filtration technique company limited product with together with the hollow fiber column of 0.45 micron for 1 micron.
2 methods
(1) the OR/80 strain of hoof-and-mouth disease seed culture of viruses poison and O/ZK/93-08 strain are inoculated respectively to the BHK21 cell of suspension culture, continued to cultivate 28 hours.
(2) results are through the foot and mouth disease virus of BHK21 cell suspension cultures respectively, and the hollow fiber column that is 5 microns, 1 micron and 0.45 micron by aperture is removed cell debris, obtains respectively the micro-filtrate of hoof-and-mouth disease seed culture of viruses poison OR/80 and O/ZK/93-08.
(3) by quality percent by volume (g/ml), in the micro-filtrate obtaining to step (2) respectively, add NaCl, the final concentration that makes NaCl is 4% (being that the NaCl quality adding in every 100ml micro-filtrate is 4g), after fully dissolving, add PEG6000, make PEG6000 final concentration for 8% (quality that is the PEG6000 that adds in every 100ml micro-filtrate is 8g), at 4 DEG C, be uniformly mixed 16h, 7000rpm high speed centrifugation 1.5h, obtain foot and mouth disease virus precipitation, use TEN buffer (0.01M Tris, 0.01M EDTA, 0.1M NaCl, PH is 7.6-8.0) resuspended virus precipitation, obtain being concentrated into 1/80 viral concentrated solution of original volume,
(4) in the ultrafiltration and concentration liquid of the micro-filtrate obtaining to step (3) respectively, add protamine sulfate, the final concentration that makes protamine sulfate is 1.5mg/ml, stirring at room temperature 1h, leave standstill after effect 0.5h, the centrifugal 45min of 7000rpm, abandons precipitation, gets centrifugal supernatant.
(5) pack sephacryl S-300 into chromatographic column, with the phosphate buffer balance chromatographic column of the 0.01M of PH7.4～8.0 of 2 times of column volumes, the concentrated foot and mouth disease virus centrifuged supernatant of centrifugal acquisition is added on respectively above gel separately, after treating that virus liquid all enters gel, then carry out eluting with the 0.01M phosphate buffer of PH7.4～8.0.
(6) collect respectively first eluting peak, (BEA adds cyclisation under the effect of sodium hydroxide of 0.2N and generates BEI after filtration sterilization, to use respectively the BEI of 2.0mM.) 30 DEG C of deactivations 28 hours, deactivation OR/80 strain and O/ZK/93-08 strain antigen mix by 1: 1 volume ratio, carry out after 7 times of dilutions with the 0.01M phosphate buffer of PH7.4～8.0, then add 206 adjuvant emulsions by 1: 1 weight ratio, subpackage obtains Schweineseuche O-shaped deactivation purified vaccine.
Embodiment 4 vaccines detect
The cattle foot and mouth disease bivalence deactivation purified vaccine (ONXC strain+JSL strain) (inner lot number: 2011RD001) that embodiment 1 is prepared is for following detection:
Attack seed culture of viruses: be O type foot and mouth disease seed culture of viruses ONXC/92 strain, the homology sodoku strain of Asia I type seed culture of viruses Asia1/JSL/GSZY/06 strain is bought from Lanzhou veterinary institute.
Control vaccine: foot and mouth disease O type, Asia I type bivalent inactivated vaccine (ONXC strain+JSL strain), product batch number: 20110202, the date of manufacture: 20110322, be Inner Mongolia Bigvet Biotechnology Co., Ltd.'s product.
Taking control vaccine as reference, carry out on the basis of physical behavior, steriling test, safety verification, efficacy test " foot and mouth disease O type, Asia I type bivalent inactivated vaccine are manufactured and inspection Trial Regulation " of drafting according to existing Ministry of Agriculture tissue, efficacy test, according to every part 1ml immunity, has increased the test of the impact on Lactation of Dairy Cow amount simultaneously.
1 physical behavior:
Cattle foot and mouth disease bivalence deactivation purified vaccine physical behavior assay is in table 1.
Table 1 physical behavior assay
2 steriling tests:
Cattle foot and mouth disease bivalence deactivation purified vaccine steriling test the results are shown in Table 2.
Table 2 steriling test result
3 safety examinations
Cattle foot and mouth disease bivalence deactivation purified vaccine safety examination the results are shown in Table 3, to the testing result of Lactation of Dairy Cow amount in table 4.
Table 3 safety examination result
The testing result of table 4 Lactation of Dairy Cow amount.
4 efficacy test cattle foot and mouth disease bivalence deactivation purified vaccine efficacy tests the results are shown in Table 5, table 6.
Table 5Asia I/JSL/GSZY/06 strain efficacy test result
Table 6ONXC/92 strain efficacy test result
5, the 146s content of cattle foot and mouth disease bivalence deactivation purified vaccine, total protein content, the inspection of DNA residual quantity
In addition we also utilize the 146s quantitative method that our company has established to carry out 146s content detection to cattle foot and mouth disease bivalence deactivation purified vaccine, and carry out protein content detection according to the lowry method of existing " Chinese Pharmacopoeia " the 3rd annex, carry out DNA content detection with ultraviolet spectrophotometry.Result is as shown in table 7.
Table 7146s content, total protein content, DNA residual quantity assay
| Vaccine lot number | 146s content | Total protein content | DNA residual quantity |
| 2011RD001 | 14.83μg/ml | 0.447mg/ml | 1.03μg/ml |
| 20110202 | 4.69μg/ml | 4.82mg/ml | 26.8μg/ml |
| Purification Seedling with contrast Seedling ratio | Improve 3.16 times | Reduce by 90.73% | Reduce by 96.16% |
Brief summary:
The foot and mouth disease virus bivalence deactivation purification Seedling that adopts this production technology to prepare, physical behavior inspection, steriling test, safety examination, efficacy test all meet the Ministry of Agriculture and draft " code " requirement.This purification Seedling is compared with the existing common Seedling that contrasts, and antigenic content improves 3.16 times; Foreign protein removes 90.73%; Nucleic acid DNA removes 96.16%, and its advantage is embodied in efficacy test result: be reduced in immunizing dose under the condition of every part of 1ml, this purification Seedling effect is all at 10 PD
50above, draft 3 PD of " code " regulation than industry portion
50at least 3 times of height.
Embodiment 5 vaccine tests
The Schweineseuche O-shaped deactivation purified vaccine (OR/80 strain+O/ZK/93-08 strain) (inner lot number: 2011RD004) that embodiment 2 is prepared is for following detection:
Attack seed culture of viruses: the homology sodoku strain that is respectively the poison OR/80 strain of pig O type foot and mouth disease and O/ZK/93-08 strain is bought from Lanzhou veterinary institute.
Control vaccine: Schweineseuche O-shaped inactivated vaccine (O/ZK/93-08 strain+OR/80 strain), product batch number: 20110104, the date of manufacture: 20110513.For Inner Mongolia Bigvet Biotechnology Co., Ltd.'s product.
Taking control vaccine as reference, carry out on the basis of physical behavior, steriling test, safety verification, efficacy test " Schweineseuche O-shaped inactivated vaccine (O/ZK/93-08 strain+OR/80 strain or OS/99 strain) is manufactured and inspection Trial Regulation " of drafting according to existing Ministry of Agriculture tissue, efficacy test is according to every part 1ml immunity.
1 physical behavior: Schweineseuche O-shaped deactivation purified vaccine physical behavior assay is in table 8.
Table 8. physical behavior assay
2 steriling tests:
Schweineseuche O-shaped deactivation purified vaccine steriling test the results are shown in Table 9.
Table 9 steriling test result
3 safety verifications: Schweineseuche O-shaped deactivation purified vaccine safety examination the results are shown in Table 10.
Table 10 safety examination result
4 efficacy tests
Schweineseuche O-shaped deactivation purified vaccine efficacy test the results are shown in Table 11, table 12.
Table 11OR/80 strain efficacy test result
Table 12O/ZK/93-08 strain efficacy test result
The 146s content of 5 Schweineseuche O-shaped deactivation purified vaccines, total protein content, the inspection of DNA residual quantity
In addition we also utilize the 146s quantitative method that our company has established to carry out 146s content detection, and according to carrying out protein content detection by the lowry method of existing " Chinese Pharmacopoeia " the 3rd annex, carrying out DNA content detection with ultraviolet spectrophotometry.The results are shown in Table 13.
Table 13.146s content, total protein content, DNA residual quantity assay
| Vaccine lot number | 146s content | Total protein content | DNA residual quantity |
| 2011RD004 | 16.94μg/ml | 0.426mg/ml | 0.972μg/ml |
| 20110104 | 5.03μg/ml | 5.43mg/ml | 24.86μg/ml |
| Purification Seedling with contrast Seedling ratio | Improve 3.37 times | Reduce by 92.15% | Reduce by 96.09% |
Brief summary:
The Schweineseuche deactivation purification Seedling that adopts this production technology to prepare, physical examination, steriling test, safety examination, efficacy test all meet the Ministry of Agriculture completely and draft " code " requirement.
This purification Seedling contrasts Seedling with existing Schweineseuche O-shaped deactivation and compares, and this purified vaccine antigenic content improves 3.37 times; Foreign protein removes 92.15%; Nucleic acid DNA removes 96.09%, and its sharpest edges are embodied in efficacy test result: be reduced in immunizing dose under the condition of every part of 1ml, this purification Seedling effect is all at 10 PD
50above, draft 3 PD of " code " regulation than industry portion
50at least 3 times of height.
The Schweineseuche O-shaped deactivation purified vaccine (OR/80 strain+O/ZK/93-08 strain) (inner lot number: 2011RD002) that embodiment 3 is prepared is for following detection:
Inspection seed culture of viruses: the homology strain that is respectively the poison OR/80 strain of pig O type foot and mouth disease and O/ZK/93-08 strain is bought from Lanzhou veterinary institute.
Control vaccine: Schweineseuche O-shaped inactivated vaccine (O/ZK/93-08 strain+OR/80 strain), product batch number: 20110104, the date of manufacture: 20110513.For Inner Mongolia Bigvet Biotechnology Co., Ltd.'s product.
Taking control vaccine as reference, carry out on the basis of physical behavior, steriling test, safety verification, efficacy test " Schweineseuche O-shaped inactivated vaccine (O/ZK/93-08 strain+OR/80 strain or OS/99 strain) is manufactured and inspection Trial Regulation " of drafting according to existing Ministry of Agriculture tissue, efficacy test is according to every part 1ml immunity.
1 physical behavior: Schweineseuche O-shaped deactivation purified vaccine physical behavior assay is in table 14.
Table 14 physical behavior assay
2 steriling tests:
Schweineseuche O-shaped deactivation purified vaccine steriling test the results are shown in Table 15.
Table 15 steriling test result
3 safety verifications: Schweineseuche O-shaped deactivation purified vaccine safety examination the results are shown in Table 16.
Table 16 safety examination result
4 efficacy tests
Schweineseuche O-shaped deactivation purified vaccine efficacy test the results are shown in Table 17, table 18.
Table 17OR/80 strain efficacy test result
Table 18O/ZK/93-08 strain efficacy test result
The 146s content of 5 Schweineseuche O-shaped deactivation purified vaccines, total protein content, the inspection of DNA residual quantity
In addition we also utilize the 146s quantitative method that our company has established to carry out 146s content detection, and according to carrying out protein content detection by the lowry method of existing " Chinese Pharmacopoeia " the 3rd annex, carrying out DNA content detection with ultraviolet spectrophotometry.The results are shown in Table 19.
Table 19.146s content, total protein content, DNA residual quantity assay
| Vaccine lot number | 146s content | Total protein content | DNA residual quantity |
| 2011RD002 | 17.15μg/ml | 0.414mg/ml | 0.966μg/ml |
| 20110104 | 5.02μg/ml | 5.426mg/ml | 24.858μg/ml |
| Purification Seedling with contrast Seedling ratio | Improve 3.42 times | Reduce by 92.37% | Reduce by 96.11% |
Brief summary:
The Schweineseuche deactivation purification Seedling that adopts this production technology to prepare, physical examination, steriling test, safety examination, efficacy test all meet the Ministry of Agriculture completely and draft " code " requirement.
This purification Seedling contrasts Seedling with existing Schweineseuche O-shaped deactivation and compares, and this purified vaccine antigenic content improves 3.42 times; Foreign protein removes 92.37%; Nucleic acid DNA removes 96.11%, and its sharpest edges are embodied in efficacy test result: be reduced in immunizing dose under the condition of every part of 1ml, this purification Seedling effect is all at 10 PD
50above, draft 3 PD of " code " regulation than industry portion
50at least 3 times of height.
The condition optimizing of embodiment 7 foot and mouth disease purified vaccine preparation methoies
1 material
1.1 vaccine strains: Asia I type poison Asia1/JSL/GSZY/06 strain is bought from Lanzhou veterinary institute.
1.2 chemical reagent: protamine sulfate is sigma company product;
1.3 chromatographic columns and gel media: be GE company product.
2 methods
According to the result of documents and materials and preliminary experiment, choose four principal elements that affect foot and mouth disease purified vaccine preparation method, be respectively the selection of protamine sulfate working concentration, protamine sulfate operative temperature and time, chromatography media, be optimized determining of condition.
2.1 protamine sulfate working concentrations are optimized
The Asia1 virus of the BHK21 cell suspension cultures of learning from else's experience, microfiltration is except broken latter concentrated 50 times, add protamine sulfate to different final concentrations, be followed successively by 0.5mg/ml, 1.0mg/ml, 1.5mg/ml, 2.0mg/ml and five Concentraton gradient of 2.5mg/ml, 4 DEG C of effects are after 1.5 hours, and 8000rpm is centrifugal, gets supernatant, detect the total protein content of supernatant, DNA content.
2.2 protamine sulfate operative temperatures and the optimization of time
The Asia1 virus of the BHK21 cell suspension cultures of learning from else's experience, microfiltration, except broken latter concentrated 50 times, adds the protamine sulfate of 1mg/ml working concentration, acts on 1.0h respectively under 4 DEG C and room temperature condition, 1.5h, 2.0h, 3.0h, 4.0h is centrifugal, gets supernatant; Detect the total protein content of supernatant, DNA content and TCID
50.
The selection of 2.3 chromatography medias
Use respectively gel media Sepharose4FF, Sepharose6FF, DEAE Sepharose, SephacrylS-500, Sephacryl S-400, the pillar of Sephacryl S-300 filling XK26, carry out balance, loading, eluting according to the characteristic of different gels, collect different eluting peaks, detect TCID
50, protein content, DNA content, calculate the antigen response rate, albumen clearance and DNA clearance.
3 results
3.1 protamine sulfate working concentrations are optimized
The Asia1 virus of the BHK21 cell suspension cultures of learning from else's experience, microfiltration, except broken latter concentrated 50 times, adds protamine sulfate to different final concentrations, is followed successively by 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2.0mg/ml and five Concentraton gradient of 2.5mg/ml, 4 DEG C of effects are after 1.5 hours, 8000rpm is centrifugal, gets supernatant, detects the total protein content of supernatant, DNA content, calculates albumen clearance and DNA clearance.Result is shown in table 20.
Table 20 protamine sulfate working concentration is optimized
According to the result of table 20, to remove after the factor such as effect and production cost of host cell foreign protein and nucleic acid at comprehensive protamine sulfate, the best working concentration of determining protamine sulfate is 1mg/ml.
3.2 protamine sulfate operative temperatures and the optimization of time
The Asia1 virus of the BHK21 cell suspension cultures of learning from else's experience, microfiltration, except broken latter concentrated 50 times, adds the protamine sulfate of 1mg/ml, acts on 1.0h respectively under 4 DEG C and room temperature condition, 1.5h, 2.0h, 3.0h, 4.0h, this time comprises stirs and the standing time, centrifugal, gets supernatant; Detect the total protein content of supernatant, DNA content and TCID
50.Result is shown in table 21.
The optimization of table 21 protamine sulfate operative temperature and time
According to the result of table 21, remove after the effect of host cell foreign protein and nucleic acid, the factor such as impact and the simplification of production technology on virus titer at comprehensive protamine sulfate, the best use of temperature and time of determining protamine sulfate is room temperature (25 DEG C) effect 1.5h.
The selection of 3.3 chromatography medias
Use respectively gel media Sepharose4FF, Sepharose6FF, DEAE Sepharose, SephacrylS-500, the pillar of Sephacryl S-400Sephacryl S-300 filling XK26, carry out balance, loading, eluting according to the characteristic of different gels, collect eluting peak, detect the TCID that collects eluent
50, protein content, DNA residual quantity, calculate the antigen response rate, albumen clearance and DNA clearance.Result is shown in table 22.Gel media Sepharose4FF, Sepharose6FF, DEAE Sepharose, Sephacryl S-500, the corresponding chromatography collection of illustrative plates of Sephacryl S-400Sephacryl S-300 is successively as shown in Fig. 1-Fig. 6
The selection of table 22 chromatography media
According to the result of table 22 and Figure of description, in Sepharose 4FF, Sepharose 6FF and DEAESepharose chromatography, foot and mouth disease virus rich region is not collected on peak at independent collection of illustrative plates, this makes the foot-and-mouth disease virus antigen response rate, albumen clearance, DNA clearance lower, does not reach the object of chromatography purification.When Sephacryl gel chromatography, foot and mouth disease virus rich region is collected on peak at first collection of illustrative plates substantially, Sephacryl S-500, Sephacryl S-400, tri-serial purification of Sephacryl S-300 have all obtained the antigen response rate and have all been greater than 50%, albumen clearance is all greater than 80%, DNA clearance and is all greater than 90% effect; But while using Sephacryl S-300 chromatography, the foot-and-mouth disease virus antigen response rate, albumen clearance and DNA clearance all obtain peak, have realized the most desired effect of chromatography purification.Comprehensive above factor, determines that the best gel filler of column chromatography is SephacrylS-300.
Claims (5)
1. a preparation method for foot and mouth disease purified vaccine, is characterized in that comprising the following steps:
(1) by the BHK21 cell of hoof-and-mouth disease seed culture of viruses poison inoculation suspension culture, continue suspension culture 15～28 hours;
(2) results, through the foot and mouth disease virus of BHK21 cell suspension cultures, are removed cell debris by microporous filter system, obtain micro-filtrate;
Described microporous filter system is that aperture is 5 microns, 1 micron uses or aperture is 5 microns with combining together with the filter element of 0.45 micron, combine use with together with the vibrosieve of 0.45 micron for 1 micron, or aperture is 5 microns, combines use for 1 micron together with 0.45 micron of hollow fiber column;
(3) micro-filtrate is concentrated into 1/10～1/100 of micro-filtrate original volume, obtains the concentrated solution of micro-filtrate;
It is concentrated or use the film bag that hollow fiber column that molecular cut off is 6kd-20kd or molecular cut off are 6kd-20kd to concentrate that described simmer down to uses PEG6000 to carry out virus precipitation;
(4) in the concentrated solution obtaining to step (3), add protamine sulfate, make the final concentration of protamine sulfate reach 1.0mg/ml～2.5mg/ml, stirring at room temperature 0.5h～2.0h, leave standstill after effect 0.5h～2.0h, centrifugal 15min～the 60min of 5000rpm～10000rpm, abandon precipitation, get centrifugal supernatant, obtain concentrated foot and mouth disease virus supernatant;
(5) pack Sephacryl gel into chromatographic column, with the phosphate buffer balance chromatographic column of the 0.01M of PH7.4～8.0 of 1～2 times of column volume, the concentrated foot and mouth disease virus supernatant of the centrifugal acquisition of step (4) is added on above Sephacryl gel, after treating that virus liquid all enters gel, then carry out eluting with the 0.01M phosphate buffer of PH7.4～8.0; Described Sephacryl gel is that component capture range is 1.0 × 10
4～1.0 × 10
8the high-resolution Sephacryl gel of globulin molecule amount;
(6) collect first eluting peak, after filtration sterilization, with the BEI30 DEG C of deactivation of 1mM～3mM 28 hours, deactivation liquid, after 2-10 doubly dilutes, added mineral oil adjuvant emulsion by 1:1 weight ratio, and subpackage obtains inactivated foot-and-mouth disease vaccine.
2. preparation method as claimed in claim 1, it is characterized in that described hoof-and-mouth disease poison strain be the each serogroup vaccine strain of foot and mouth disease virus wherein at least one.
3. preparation method as claimed in claim 1, is characterized in that described high-resolution Sephacryl gel component capture range is 1.0 × 10
4～1.5 × 10
6globulin molecule amount.
4. the foot and mouth disease purified vaccine being prepared by the preparation method described in claim 1-3 any one.
5. the application of foot and mouth disease purified vaccine claimed in claim 4 in preparation prevention animal foot and mouth disease disease medicament.
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| CN109387582B (en) * | 2017-09-06 | 2020-02-21 | 申联生物医药（上海）股份有限公司 | Quantitative method of foot-and-mouth disease antigen |
| CN108042798B (en) * | 2017-12-22 | 2020-10-16 | 吉林冠界生物技术有限公司 | Method for producing recombinant avian influenza virus inactivated vaccine by using suspension cells |
| CN114540317A (en) * | 2022-03-17 | 2022-05-27 | 新疆方牧生物科技有限公司 | Suspension culture process for inactivating orohoof virus |
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| CN1533242A (en) * | 2001-04-26 | 2004-09-29 | ��������ũҵ�� | Foot and mouth disease virus vaccine |
| CN101130075A (en) * | 2007-07-23 | 2008-02-27 | 中国农业科学院兰州兽医研究所 | Tervalence inactivated vaccine for type O, A, Asia-1 hoof-and-mouth disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1533242A (en) * | 2001-04-26 | 2004-09-29 | ��������ũҵ�� | Foot and mouth disease virus vaccine |
| CN101130075A (en) * | 2007-07-23 | 2008-02-27 | 中国农业科学院兰州兽医研究所 | Tervalence inactivated vaccine for type O, A, Asia-1 hoof-and-mouth disease |
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| Title |
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| 硫酸鱼精蛋白去除Vero细胞乙脑疫苗残余DNA方法的条件优化;韩德胜等;《微生物学免疫学进展》;20071130;第35卷(第4期);14-18 * |
| 韩德胜等.硫酸鱼精蛋白去除Vero细胞乙脑疫苗残余DNA方法的条件优化.《微生物学免疫学进展》.2007,第35卷(第4期),14-18. |
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